Individuals with hemophilia or taking anticoagulants are at risk for bleeding, but where they bleed is different. Understanding how these two types of perturbations to the hemostatic system interact in distinct vascular beds (VBs) will inform decisions about bleeding treatment. Bleeding is treated using prohemostatic agents, but individual responses to these agents are highly variable and the mechanisms underlying the variability are unknown. Hemostasis is a nonlinear process involving complex coagulation biochemistry coupled to platelet function, VBs, and biophysical mechanisms including blood flow; it is well suited for study with an integrated computational and experimental approach. The long-term goal of this research is to develop mathematical models that improve the treatment of bleeding. The overall objective is to develop and validate mathematical models of bleeding that will identify mechanisms underlying variable responses to prohemostatics and in different VBs. The central hypothesis is that global sensitivity analysis (GSA) applied to mechanistic mathematical models of bleeding will elucidate synergies and/or cooperation among platelet, vascular, and plasma components and predict experimentally-verified hemostatic responses. This hypothesis is based on preliminary data produced using exactly this approach in the applicants? laboratories. The rationale is that the proposed quantitative methods and the identification of modifiers of the hemostatic response will together provide a foundation for developing assays that test for specific and previously unidentified biomarkers. Guided by strong preliminary data, this hypothesis will be tested in three specific aims: 1) Develop and refine mathematical models of hemostasis, 2) Determine the mechanistic link between bleeding site and bleeding cause, and 3) Identify modifiers of hemostasis that regulate responses to prohemostatics in hemophilia A. In Aim 1, existing models will be extended to include essential features of platelet and fibrin dynamics and validated with microfluidic assays. In Aim 2, submodels of anticoagulants will be developed and incorporated into the hemostasis models. Experimental measurements of VB characteristics will be acquired. GSA will identify the causes of VB site-specific variability in the hemostatic response. In Aim 3, submodels of prohemostatics will be developed and incorporated into the hemostasis models. GSA will identify the causes of variability in responses to them during treatment of hemophilia A. The approach is innovative because (1) the mathematical models and experimental assays will be developed in tandem to iteratively and optimally inform one another, and (2) novel submodels of anticoagulants and prohemostatics will be added to a comprehensive model of the hemostatic system that includes platelet, fibrin, and VB dynamics coupled to coagulation and flow. The proposed research is significant because it is expected to (1) provide mechanistic explanations for site- specific bleeding in hemophilia A and anticoagulant use, and (2) provide mechanism-based knowledge to potentially guide clinical decisions in the treatment of bleeding.